The link protein-like sequence of CLEVER-1 does not mediate hyaluronan binding. (A) Peripheral lymph node sections were pretreated with 200 μg/mL hyaluronan (HA) or left untreated (PBS). Thereafter the sections were stained for CLEVER-1 expression using mAbs 3-266 and 3-372. Arrows point to HEV; L indicates lymphatic endothelium. Bar, 50 μm. (B) CLEVER-1 and control proteins were immunoaffinity purified from human peripheral lymph node lysate. Silver staining shows the purity of the isolated endogenous CLEVER-1 protein (arrow). MW indicates molecular weight standards in kilodaltons. (C) CD4 receptor globulin (CD4-Ig), CD44 receptor globulin (CD44-Ig), BSA, CLEVER-1 protein, and proteins from a control eluate (control) were absorbed on the bottom of an ELISA plate. The immobilization of the relevant proteins was confirmed by sequential incubation of the wells with anti-CD44 mAb Hermes-3 (H-3), anti-CLEVER-1 mAb 3-372, or a negative control mAb. The hyaluronan binding capacity of the bound proteins was analyzed by their ability to bind FITC-labeled hyaluronan (HA). The results from a representative experiment (2 wells for each treatment) are shown as the absolute fluorescent absorbances (mean ± SEM). Similar results were obtained in 2 other independent experiments.